Dataset Information

Quantitative proteomics of intracellular Porphyromonas gingivalis.

ABSTRACT: Whole-cell quantitative proteomic analyses were conducted to investigate the change from an extracellular to intracellular lifestyle for Porphyromonas gingivalis, a Gram-negative intracellular pathogen associated with periodontal disease. Global protein abundance data for P. gingivalis strain ATCC 33277 internalized for 18 h within human gingival epithelial cells and controls exposed to gingival cell culture medium were obtained at sufficient coverage to provide strong evidence that these changes are profound. A total of 385 proteins were overexpressed in internalized P. gingivalis relative to controls; 240 proteins were shown to be underexpressed. This represented in total about 28% of the protein encoding ORFs annotated for this organism, and slightly less than half of the proteins that were observed experimentally. Production of several proteases, including the classical virulence factors RgpA, RgpB, and Kgp, was decreased. A separate validation study was carried out in which a 16-fold dilution of the P. gingivalis proteome was compared to the undiluted sample in order to assess the quantitative false negative rate (all ratios truly alternative). Truly null (no change) abundance ratios from technical replicates were used to assess the rate of quantitative false positives over the entire proteome. A global comparison between the direction of abundance change observed and previously published bioinformatic gene pair predictions for P. gingivalis will assist with future studies of P. gingivalis gene regulation and operon prediction.

SUBMITTER: Xia Q

PROVIDER: S-EPMC2660851 | biostudies-literature | 2007 Dec

REPOSITORIES: biostudies-literature

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Quantitative proteomics of intracellular Porphyromonas gingivalis.

Xia Qiangwei Q Wang Tiansong T Taub Fred F Park Yoonsuk Y Capestany Cindy A CA Lamont Richard J RJ Hackett Murray M

Proteomics 20071201 23

Whole-cell quantitative proteomic analyses were conducted to investigate the change from an extracellular to intracellular lifestyle for Porphyromonas gingivalis, a Gram-negative intracellular pathogen associated with periodontal disease. Global protein abundance data for P. gingivalis strain ATCC 33277 internalized for 18 h within human gingival epithelial cells and controls exposed to gingival cell culture medium were obtained at sufficient coverage to provide strong evidence that these change ...[more]

PMID: 17979175

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Project description:Porphyromonas gingivalis is a Gram-negative intracellular pathogen associated with periodontal disease. We have previously reported on whole-cell quantitative proteomic analyses to investigate the differential expression of virulence factors as the organism transitions from an extracellular to intracellular lifestyle. The original results with the invasive strain P. gingivalis ATCC 33277 were obtained using the genome sequence available at the time, strain W83 [GenBank: AE015924]. We present here a re-processed dataset using the recently published genome annotation specific for strain ATCC 33277 [GenBank: AP009380] and an analysis of differential abundance based on metabolic pathways rather than individual proteins.Qualitative detection was observed for 1266 proteins using the strain ATCC 33277 annotation for 18 hour internalized P. gingivalis within human gingival epithelial cells and controls exposed to gingival cell culture medium, an improvement of 7% over the W83 annotation. Internalized cells showed increased abundance of proteins in the energy pathway from asparagine/aspartate amino acids to ATP. The pathway producing one short chain fatty acid, propionate, showed increased abundance, while that of another, butyrate, trended towards decreased abundance. The translational machinery, including ribosomal proteins and tRNA synthetases, showed a significant increase in protein relative abundance, as did proteins responsible for transcription.Use of the ATCC 33277 specific genome annotation resulted in improved proteome coverage with respect to the number of proteins observed both qualitatively in terms of protein identifications and quantitatively in terms of the number of calculated abundance ratios. Pathway analysis showed a significant increase in overall protein synthetic and transcriptional machinery in the absence of significant growth. These results suggest that the interior of host cells provides a more energy rich environment compared to the extracellular milieu. Shifts in the production of cytotoxic fatty acids by intracellular P. gingivalis may play a role in virulence. Moreover, despite extensive genomic re-arrangements between strains W83 and 33277, there is sufficient sequence similarity at the peptide level for proteomic abundance trends to be largely accurate when using the heterologous strain annotated genome as the reference for database searching.

Project description:The asaccharolytic anaerobe Porphyromonas gingivalis metabolizes proteins it encounters in the periodontal pocket, including host-derived glycoproteins such as mucins and immunoglobulins. Often, these proteins are protected by a diverse array of carbohydrates tethered to the polypeptide chain via glycolytic bonds, and P. gingivalis produces enzymes capable of liberating these carbohydrates, exposing the proteinaceous core. In this study, we investigated the effect of individual monosaccharides, including galactose, l-fucose, mannose, and glucose, on the growth and physiology of P. gingivalis Of the carbohydrates tested, only galactose noticeably altered the density of the bacterial culture, and we observed that cultures grown with galactose reached significantly higher densities during stationary phase. Importantly, electron micrographs and plating of P. gingivalis in stationary phase demonstrated that the presence of galactose did not increase cell numbers; instead, the higher densities resulted from the expansion of individual cells which contained large intracellular granules. Initial attempts to characterize these granules revealed only a subtle increase in soluble carbohydrates, suggesting they are likely not composed of stored carbohydrate. Also, an analysis of major surface polysaccharides via an enzyme-linked immunosorbent assay (ELISA) did not reveal significant differences between cells grown with or without galactose. Finally, an initial investigation of the transcriptional changes elicited by galactose in late exponential phase suggested that genes important for cell shape and for the general stress response may play roles in this phenomenon. Overall, galactose, a monosaccharide commonly present on the surfaces of host proteins, substantially alters the physiology of P. gingivalis via the production of large, currently undefined, intracellular granules.IMPORTANCE Environmental perturbations are central to the ability of pathobionts, such as Porphyromonas gingivalis, to promote the development of diseased sites. In the case of periodontal disease, increased local pH, a shift to anaerobic surroundings, and the accumulation of Gram-negative anaerobes at the expense of Gram-positive cocci are known ecological fluctuations prominently associated with progression toward disease. Importantly, in contrast, the alterations to subgingival food webs in disease sites remain poorly characterized. We hypothesized that given the dramatic shift in community structure during disease, it is possible that free carbohydrates, which would typically be readily metabolized by Gram-positive cocci after cleavage from glycoproteins, may increase in concentration locally and thereby affect the physiological state of the subgingival microbiota. In this study, we explored the impact of free monosaccharides on P. gingivalis to gain deeper insight into the effect of dysbiotic conditions on the growth and physiology of this periodontal pathogen.

Dataset Information

Quantitative proteomics of intracellular Porphyromonas gingivalis.

Publications

Quantitative proteomics of intracellular Porphyromonas gingivalis.

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